The productive phase of the HPV life cycle is restricted to the uppermost layer of the epithelium, in cells that have normally exited the cell cycle. The E7 protein alters cell cycle regulation to push differentiating cells back into the cell cycle, allowing for productive replication and virion production. Cell cycle deregulation by E7 leads to genomic instability that is a driving force in cancer development. Our long-term goal is to understand mechanisms that regulate productive viral replication, which is important to understanding how HPV causes cancer. HPV genomes are histone-associated, though the impact of histone post-translational modifications on the viral life cycle is unclear. This proposal focuses on understanding how HPV utilizes the SETD2 methyltransferase to facilitate viral replication. SETD2 adds the trimethyl mark to histone H3 on lysine 36 (H3K36me3) during transcription elongation and regulates multiple cellular processes through the recruitment of numerous effector proteins to H3K36me3. We will identify the mechanisms by which SETD2 activity contributes to viral replication and determine how E7 increases the stability of SETD2. We hypothesize that HPV epigenetically regulates the viral life cycle through SETD2-mediated H3K36me3 on viral, as well as cellular chromatin. Specific Aims to test this are: (1) To determine the mechanism by which SETD2 facilitates viral replication by examining whether SETD2 knockdown affects transcription, RNA processing and DNA repair on viral genomes, as well as by identifying H3K36me3 readers bound to HPV DNA. (2) To determine the mechanism by which E7 increases the protein stability of SETD2 by examining known modulators of SETD2 stability, as well as by identifying SETD2 interacting partners though a non-biased approach using a BirA- tagged SETD2 coupled with mass spectrometry. Since SETD2 is essential to the maintenance of genomic stability, it is important to understand how HPV utilizes this epigenetic modifier to facilitate viral replication. Understanding how SETD2 activity promotes the viral life cycle will provide insight into mechanisms of viral persistence, as well as genomic instability. These studies may also identify therapeutic targets for the treatment of HPV-associated diseases.